Dynamics of SNARE Assembly and Disassembly during Sperm Acrosomal Exocytosis

The dynamics of SNARE assembly and disassembly during membrane recognition and fusion is a central issue in intracellular trafficking and regulated secretion. Exocytosis of sperm's single vesicle—the acrosome—is a synchronized, all-or-nothing process that happens only once in the life of the cell and depends on activation of both the GTP-binding protein Rab3 and of neurotoxin-sensitive SNAREs. These characteristics make acrosomal exocytosis a unique mammalian model for the study of the different phases of the membrane fusion cascade. By using a functional assay and immunofluorescence techniques in combination with neurotoxins and a photosensitive Ca(2+) chelator we show that, in unactivated sperm, SNAREs are locked in heterotrimeric cis complexes. Upon Ca(2+) entry into the cytoplasm, Rab3 is activated and triggers NSF/α-SNAP-dependent disassembly of cis SNARE complexes. Monomeric SNAREs in the plasma membrane and the outer acrosomal membrane are then free to reassemble in loose trans complexes that are resistant to NSF/α-SNAP and differentially sensitive to cleavage by two vesicle-associated membrane protein (VAMP)–specific neurotoxins. Ca(2+) must be released from inside the acrosome to trigger the final steps of membrane fusion that require fully assembled trans SNARE complexes and synaptotagmin. Our results indicate that the unidirectional and sequential disassembly and assembly of SNARE complexes drive acrosomal exocytosis

Sistemas inteligentes para la evaluación de la calidad de la información en la web

En este artículo se describen, en forma resumida, los trabajos de investigación y desarrollo que se están llevando a cabo en la línea de investigación “Sistemas Inteligentes” en las áreas de Text Mining, Web Mining y Web Intelligence, y que abordan principalmente tareas como: clustering de textos cortos multilingües, categorización semántica de textos, medidas de calidad de la información en la Web, detección de plagio y atribución de autoría, minería de opinión y sentimientos, integración de agentes y técnicas de minería de textos, y uso de arquitecturas cognitivas para agentes web; en especial aquellas basadas en lógica, razonamiento argumentativo y teoría de decisión cualitativa. En particular, pondremos especial énfasis en aquellas problemáticas que se están comenzando a investigar en forma conjunta con investigadores de Alemania, Austria, España y Grecia en el contexto de un proyecto FP7, recientemente aprobado en la Unión Europea.Eje: Agentes y sistemas inteligentesRed de Universidades con Carreras en Informática (RedUNCI

Coinfection with Different Trypanosoma cruzi Strains Interferes with the Host Immune Response to Infection

A century after the discovery of Trypanosoma cruzi in a child living in Lassance, Minas Gerais, Brazil in 1909, many uncertainties remain with respect to factors determining the pathogenesis of Chagas disease (CD). Herein, we simultaneously investigate the contribution of both host and parasite factors during acute phase of infection in BALB/c mice infected with the JG and/or CL Brener T. cruzi strains. JG single infected mice presented reduced parasitemia and heart parasitism, no mortality, levels of pro-inflammatory mediators (TNF-α, CCL2, IL-6 and IFN-γ) similar to those found among naïve animals and no clinical manifestations of disease. On the other hand, CL Brener single infected mice presented higher parasitemia and heart parasitism, as well as an increased systemic release of pro-inflammatory mediators and higher mortality probably due to a toxic shock-like systemic inflammatory response. Interestingly, coinfection with JG and CL Brener strains resulted in intermediate parasitemia, heart parasitism and mortality. This was accompanied by an increase in the systemic release of IL-10 with a parallel increase in the number of MAC-3+ and CD4+ T spleen cells expressing IL-10. Therefore, the endogenous production of IL-10 elicited by coinfection seems to be crucial to counterregulate the potentially lethal effects triggered by systemic release of pro-inflammatory mediators induced by CL Brener single infection. In conclusion, our results suggest that the composition of the infecting parasite population plays a role in the host response to T. cruzi in determining the severity of the disease in experimentally infected BALB/c mice. The combination of JG and CL Brener was able to trigger both protective inflammatory immunity and regulatory immune mechanisms that attenuate damage caused by inflammation and disease severity in BALB/c mice

Cyclooxygenase-2 and prostaglandin E<inf>2</inf> signaling through prostaglandin receptor EP- 2 favor the development of myocarditis during acute trypanosoma cruzi infection

Inflammation plays an important role in the pathophysiology of Chagas disease, caused by Trypanosoma cruzi. Prostanoids are regulators of homeostasis and inflammation and are produced mainly by myeloid cells, being cyclooxygenases, COX-1 and COX-2, the key enzymes in their biosynthesis from arachidonic acid (AA). Here, we have investigated the expression of enzymes involved in AA metabolism during T. cruzi infection. Our results show an increase in the expression of several of these enzymes in acute T. cruzi infected heart. Interestingly, COX-2 was expressed by CD68+ myeloid heart-infiltrating cells. In addition, infiltrating myeloid CD11b+Ly6G- cells purified from infected heart tissue express COX-2 and produce prostaglandin E2 (PGE2) ex vivo. T. cruzi infections in COX-2 or PGE2- dependent prostaglandin receptor EP-2 deficient mice indicate that both, COX-2 and EP-2 signaling contribute significantly to the heart leukocyte infiltration and to the release of chemokines and inflammatory cytokines in the heart of T. cruzi infected mice. In conclusion, COX-2 plays a detrimental role in acute Chagas disease myocarditis and points to COX-2 as a potential target for immune intervention.This work was supported by (NG) grants from “Fondo de Investigaciones Sanitarias” (PS09/00538 and PI12/00289); “Universidad Autónoma de Madrid” and “Comunidad de Madrid” (CC08-UAM/SAL-4440/08); by (MF) grants from “Ministerio de Ciencia e Innovación” (SAF2010-17833); “Red de Investigación de Centros de Enfermedades Tropicales” (RICET RD12/0018/0004); European Union (HEALTH-FE-2008-22303, ChagasEpiNet); AECID Cooperation with Argentine (A/025417/09 and A/031735/10), Comunidad de Madrid (S-2010/BMD- 2332) and “Fundación Ramón Areces”. NAG was recipient of a ISCIII Ph.D. fellowship financed by the Spanish “Ministerio de Sanidad”. CCM and HC were recipients of contracts from SAF2010-17833 and PI060388, respectively.Peer Reviewe

Model inter-comparison on crop rotation effects ? an intermediate report

Data of diverse crop rotations from five locations across Europe were distributed to modelers to investigate the capability of models to handle complex crop rotations and management interactions

Abiraterone acetate plus prednisolone with or without enzalutamide for patients with metastatic prostate cancer starting androgen deprivation therapy: final results from two randomised phase 3 trials of the STAMPEDE platform protocol

Background: Abiraterone acetate plus prednisolone (herein referred to as abiraterone) or enzalutamide added at the start of androgen deprivation therapy improves outcomes for patients with metastatic prostate cancer. Here, we aimed to evaluate long-term outcomes and test whether combining enzalutamide with abiraterone and androgen deprivation therapy improves survival. Methods: We analysed two open-label, randomised, controlled, phase 3 trials of the STAMPEDE platform protocol, with no overlapping controls, conducted at 117 sites in the UK and Switzerland. Eligible patients (no age restriction) had metastatic, histologically-confirmed prostate adenocarcinoma; a WHO performance status of 0–2; and adequate haematological, renal, and liver function. Patients were randomly assigned (1:1) using a computerised algorithm and a minimisation technique to either standard of care (androgen deprivation therapy; docetaxel 75 mg/m2 intravenously for six cycles with prednisolone 10 mg orally once per day allowed from Dec 17, 2015) or standard of care plus abiraterone acetate 1000 mg and prednisolone 5 mg (in the abiraterone trial) orally or abiraterone acetate and prednisolone plus enzalutamide 160 mg orally once a day (in the abiraterone and enzalutamide trial). Patients were stratified by centre, age, WHO performance status, type of androgen deprivation therapy, use of aspirin or non-steroidal anti-inflammatory drugs, pelvic nodal status, planned radiotherapy, and planned docetaxel use. The primary outcome was overall survival assessed in the intention-to-treat population. Safety was assessed in all patients who started treatment. A fixed-effects meta-analysis of individual patient data was used to compare differences in survival between the two trials. STAMPEDE is registered with ClinicalTrials.gov (NCT00268476) and ISRCTN (ISRCTN78818544). Findings: Between Nov 15, 2011, and Jan 17, 2014, 1003 patients were randomly assigned to standard of care (n=502) or standard of care plus abiraterone (n=501) in the abiraterone trial. Between July 29, 2014, and March 31, 2016, 916 patients were randomly assigned to standard of care (n=454) or standard of care plus abiraterone and enzalutamide (n=462) in the abiraterone and enzalutamide trial. Median follow-up was 96 months (IQR 86–107) in the abiraterone trial and 72 months (61–74) in the abiraterone and enzalutamide trial. In the abiraterone trial, median overall survival was 76·6 months (95% CI 67·8–86·9) in the abiraterone group versus 45·7 months (41·6–52·0) in the standard of care group (hazard ratio [HR] 0·62 [95% CI 0·53–0·73]; p&lt;0·0001). In the abiraterone and enzalutamide trial, median overall survival was 73·1 months (61·9–81·3) in the abiraterone and enzalutamide group versus 51·8 months (45·3–59·0) in the standard of care group (HR 0·65 [0·55–0·77]; p&lt;0·0001). We found no difference in the treatment effect between these two trials (interaction HR 1·05 [0·83–1·32]; pinteraction=0·71) or between-trial heterogeneity (I2 p=0·70). In the first 5 years of treatment, grade 3–5 toxic effects were higher when abiraterone was added to standard of care (271 [54%] of 498 vs 192 [38%] of 502 with standard of care) and the highest toxic effects were seen when abiraterone and enzalutamide were added to standard of care (302 [68%] of 445 vs 204 [45%] of 454 with standard of care). Cardiac causes were the most common cause of death due to adverse events (five [1%] with standard of care plus abiraterone and enzalutamide [two attributed to treatment] and one (&lt;1%) with standard of care in the abiraterone trial). Interpretation: Enzalutamide and abiraterone should not be combined for patients with prostate cancer starting long-term androgen deprivation therapy. Clinically important improvements in survival from addition of abiraterone to androgen deprivation therapy are maintained for longer than 7 years. Funding: Cancer Research UK, UK Medical Research Council, Swiss Group for Clinical Cancer Research, Janssen, and Astellas

Syntaxin1A Is Assembled in Toxin-Resistant Complexes That Are Disassembled by NSF/α-SNAP or by Sperm Activation

<div><p>(A) Permeabilized spermatozoa were incubated for 15 min at 37 °C with increasing concentrations of BoNT/C (black circles, wild type; grey circles, EA, a protease-inactive mutant) and then stimulated with 10 μM Ca²⁺ for 15 min at 37 °C. Afterwards, sperm were fixed and AE measured as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030323#s4" target="_blank">Materials and Methods</a>.</p> <p>(B) To assess the assembly state of syntaxin1A, sperm were incubated with 100 nM BoNT/C (15 min at 37 °C), and the cells were then fixed and immunostained with an anti-syntaxin1A antibody recognizing an epitope that is cleaved by the toxin. To prevent AE, which would release syntaxin into the medium by vesiculation of the acrosome, intra-acrosomal Ca²⁺ was chelated with 10 μM BAPTA-AM (15 min at 37 °C, B-AM). The toxin treatment in resting sperm (BoNT/C) or B-AM-loaded sperm (B-AM→BoNT/C) had no effect on the syntaxin labeling compared to untreated sperm (control). However, when 310 nM NSF and 500 nM α-SNAP were added to the system to promote the disassembly of SNARE complexes, the toxin significantly decreased the syntaxin labeling (B-AM→NSF/αS→BoNT/C). The BoNT/C treatment also affected syntaxin labeling when sperm were stimulated for 15 min at 37 °C with 10 μM free Ca²⁺ (B-AM→ BoNT/C→Ca²⁺) or 300 nM Rab3A (B-AM→BoNT/C→Rab3A). The protease-inactive mutant did not affect labeling under these conditions (B-AM→BoNT/C-EA→Ca²⁺). Fluorescence was normalized as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030323#s4" target="_blank">Materials and Methods</a>. The data represent the mean ± SEM. Statistical analysis is provided in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030323#st005" target="_blank">Table S5</a>.</p></div

Effect of BoNT/C on Syntaxin1A Immunofluorescence

<p>Sperm were incubated with 100 nM BoNT/C (15 min at 37 °C) as explained in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030323#pbio-0030323-g004" target="_blank">Figure 4</a>. The cells were then fixed and triple-stained with an anti-syntaxin1A antibody that recognizes an epitope trimmed by the toxin (red; [A, D, G, and J]), FITC-PSA to differentiate between reacted and intact sperm (green; [B, E, H, and K]), and Hoechst 33258 to visualize all cells in the field (blue; [C, F, I, and L]). Notice that spontaneously reacted sperm were negative for syntaxin1A staining (arrowheads in [D] and [E]). BoNT/C had no effect on resting sperm (compare [A–C] with [D–F]). However, labeling in sperm stimulated with 10 μM Ca²⁺ in the presence of BAPTA-AM to prevent exocytosis (observe that PSA staining is not affected) was significantly reduced by the toxin (asterisks, [G]). In contrast, the same experimental condition in the presence of the protease-inactive toxin (BoNT/C-EA) had no effect (J–L). Bars = 5 μm.</p

VAMP2 Is Engaged in Loose SNARE Complexes before the Efflux of Intra-Acrosomal Ca²⁺

<p>Permeabilized spermatozoa were loaded with 10 μM BAPTA-AM (B-AM) for 15 min at 37 °C to chelate intra-acrosomal Ca²⁺. AE was then initiated by adding 10 μM free Ca²⁺ (Ca²⁺). After 15 min incubation at 37 °C to allow exocytosis to proceed to the intra-acrosomal Ca²⁺-sensitive step, 100 nM neurotoxins recognizing VAMP (BoNT/B or TeTx) were added to the tubes and the samples were incubated for 15 min at 37 °C (B-AM→Ca²⁺→neurotoxin, black bars). Samples were then immunolabeled with an anti-VAMP2 antibody as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030323#s4" target="_blank">Materials and Methods</a>. Notice that at this stage VAMP2 immunolabeling was sensitive to BoNT/B but not to TeTx. Several other conditions are included (grey bars). The toxins did not affect VAMP2 staining in resting sperm (compare control versus B-AM→neurotoxin). However, the toxins decreased the VAMP2 labeling when present during stimulation (B-AM→neurotoxin→Ca²⁺). Fluorescence was normalized as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030323#s4" target="_blank">Materials and Methods</a> (mean ± SEM). Statistical analysis is provided in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030323#st008" target="_blank">Table S8</a>.</p

SNAREs Reassemble in Loose Complexes That Are Resistant to NSF/α-SNAP before the Efflux of Intra-Acrosomal Ca²⁺

<div><p>(A) Permeabilized spermatozoa were loaded with 10 μM NP-EGTA-AM (NP) for 15 min at 37 °C to chelate intra-acrosomal Ca²⁺. AE was then initiated by adding 10 μM free Ca²⁺ (Ca²⁺). After 15 min incubation at 37 °C to allow exocytosis to proceed to the intra-acrosomal Ca²⁺-sensitive step, 800 nM recombinant SNAP25 (SNAP25) was added to compete with endogenous SNAP25. Intra-acrosomal Ca²⁺ was replenished by photolysis of NP-EGTA-AM (hν), and the samples were incubated for 5 min to promote exocytosis (NP→Ca²⁺→SNAP25→hν, black bar). Sperm were then fixed and AE was measured as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030323#s4" target="_blank">Materials and Methods</a>.</p> <p>(B) Permeabilized spermatozoa were loaded with 10 μM NP-EGTA-AM (NP) for 15 min at 37 °C. AE was then initiated by adding 10 μM free Ca²⁺ (Ca²⁺) or 300 nM Rab3A (Rab3A). After 15 min incubation at 37 °C, 100 nM neurotoxin recognizing VAMP (BoNT/B and TeTx) was added to the tubes to assess whether the SNAREs had reassembled in loose <i>trans</i> complexes sensitive to BoNT/B but not to TeTx. After 15 min incubation at 37 °C, intra-acrosomal Ca²⁺ was replenished by photolysis of NP-EGTA-AM (hν), and the samples were incubated for 5 min to promote exocytosis (NP→Ca²⁺/Rab3A→neurotoxin→hν, black bars). Sperm were then fixed and AE measured as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030323#s4" target="_blank">Materials and Methods</a>.</p> <p>(C) To assess whether NSF/α-SNAP can disassemble loose <i>trans</i> SNARE complexes, permeabilized sperm treated as in (B) were incubated with TeTx in the presence of 310 nM NSF and 500 nM α-SNAP (NP→Ca²⁺/Rab3A→NSF/αS+TeTx→hν, black bars).</p> <p>Several controls were included in (A), (B), and (C) (grey bars): background AE in the absence of any stimulation (control); AE stimulated by 10 μM free Ca²⁺ (Ca²⁺) or 300 nM Rab3A (Rab3A); inhibitory effect of NP-EGTA-AM in the dark (NP→Ca²⁺/Rab3A→dark) and the recovery upon illumination (NP→Ca²⁺/Rab3A→hν); inhibitory effect when SNAP25 was present throughout the incubations (NP→SNAP25→Ca²⁺→hν); inhibitory effect when the neurotoxins were present throughout the incubations (NP→neurotoxin→Ca²⁺/Rab3A→hν); and the effect of NSF/α-SNAP on SNARE complexes in unstimulated sperm (NSF/αS+TeTx→TPEN→Rab3A→hν). The data were normalized as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030323#s4" target="_blank">Materials and Methods</a> (mean ± SEM). Statistical analysis is provided in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030323#st007" target="_blank">Table S7</a>.</p></div